EP1369120B1 - Treatment of flavivirus infection - Google Patents

Treatment of flavivirus infection Download PDF

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EP1369120B1
EP1369120B1 EP03012517A EP03012517A EP1369120B1 EP 1369120 B1 EP1369120 B1 EP 1369120B1 EP 03012517 A EP03012517 A EP 03012517A EP 03012517 A EP03012517 A EP 03012517A EP 1369120 B1 EP1369120 B1 EP 1369120B1
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virus
use according
effective amount
flaviviridae
ribavirin
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French (fr)
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EP1369120A1 (en
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Tsu-An Hsu
Yuan-Chin Tsai
Der-Ren Hwang
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National Health Research Institutes
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/245—Bismuth; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Flaviviridae is a family of enveloped, positive single-stranded RNA viruses. It includes hepatitis C virus ("HCV”), GB virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, and yellow fever virus. Flaviviridae infection is a worldwide health problem.
  • HCV hepatitis C virus
  • GB virus GB virus
  • dengue virus Japanese encephalitis virus
  • St. Louis encephalitis virus St. Louis encephalitis virus
  • West Nile virus and yellow fever virus.
  • Flaviviridae infection is a worldwide health problem.
  • HCV infection is estimated to affect 170 million people (WHO (1997) Wkly Epidemiol Rec. 72:341-344). Patients with liver damage resulting from HCV infection may develop chronic liver diseases, such as cirrhosis and hepatocellular carcinoma (Bartenschlager & Lohmann (2000) J Gen Virol . 81:1631-1648). HCV infection has been treated with a combination of interferon ⁇ and ribavirin (1- ⁇ -D-ribofuranosyl-1,2,4-triazole-3-carboxamide). See, e.g., Foster & Thomas (2000) Baillieres Best Pract Res Clin Gastroenterol. 14:255-64.
  • WO9511033 discloses pharmaceutical compositions containing polyoxometallates and pharmaceutically acceptable derivatives thereof. The use of such compounds or compositions in therapy for the treatment or prophylaxis of infections by viruses which are confirmed or probable members of the family Flaviviridae including Hepatitis C.
  • EPI 108359 relates to a biocidal material comprising, as an active ingredient, a compound represented by General Formula (Ag2O)a(A2O)b(BO)c(C2O3)d(SiO2)e(DO2)f(E2O5)g
  • A represents at least one species selected from the group consisting of alkali metallic elements, Cu, H and ammonium
  • B is at least one species selected from the group consisting of Fe, Cu, Zn, alkali earth metallic element, Ni, Mn, Co, Cd, Hg and Au
  • C represents at least one species selected from the group consisting of Fe, Al, Mn, B, Co, Cr, V, Sc, Y, La, Ga, In, Sb and Bi
  • D represents at least one species selected from the group consisting of Ce, Mn, C, Hf and Os
  • E represents at least one member selected from P, Sb, V, Nb, Ta and Bi
  • the subscriptions respectively are numbers which satisfy 0 ⁇ a, O ⁇ a+b
  • WO02096412 disclose various embodiments directed to pentavalent antimonials, purified pentavalent antimonials, and pentavalent antimonials that are comprised of an antimonial portion and an organic moeity portion.
  • agents and methods of screening agents which mimic the activity of pentavalent antimonials, particularly sodium stibogluconate and glucatime.
  • An appropriate organic moeity may be selected based upon a desired interaction (e.g. steric action) with active site of a cellular component (e.g. a PTPase).
  • the active site includes a cysteine residue which may be impacted by the disclosed compositions.
  • US2003039702 disclose a compound for use as an antiviral drug, mainly containing salts of heteropolyanions consisting of a tungstoantimonate (III) vanadium-mixed metal oxide or related salts represented by formula [(XW9O33)2V3O3]p-, where p is a positive number between 9 and 12 and X is Sb, P. As or Bi and especially Sb.
  • An antiviral drug having a broad spectrum of antiviral activity, high potent efficacy and low toxicity is provided.
  • This invention is based, in part, on use of arsenic-, antimony-, or bismuth-containing compounds for the preparation of therapeutic agents for treating flaviviridae infection.
  • a method for treating infection by a flaviviridae virus includes administering to a subject in need thereof (including a subject identified as in need of such treatment) an effective amount of a compound of the formula: A w B x C y D z , in which A is Li, Na, K, Rb, or Cs; B is As, Sb, or Bi; C is an oxygen or sulfur atom; D is a monodentate ligand, a bidentate ligand, or a tridentate ligand; w is 0, 1, 2, or 3; x is 1, 2, 3, or 4; y is 0, 1, 2, 3, 4, or 5; z is 0, 1, 2, 3, 4, or 5; and provided that at least one of y and z is not 0.
  • stable refers to a compound which possesses stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).
  • Sb, and Bi delineated herein include bivalent, trivalent, and pentavalent forms.
  • a subset of the compounds covered by the formula described above is featured by that each of w and z is 0.
  • x is 2 and y is 3.
  • Exemplary compounds include As 2 O 3 , Sb 2 O 3 , and Bi 2 O 3 .
  • x is 2 or 4, and y is 2, 4 or 5, or x is 1 and y is 1.
  • Exemplary compounds include As 2 O 5 , AsS, As 2 S 2 , and As 4 S 4 .
  • Another subset of the compounds is featured by that w is 1, 2, or 3 and z is 0.
  • w is 1 and y is 2.
  • An exemplary compound is NaAsO 2 .
  • a third subset of the compounds is featured by that y is 0.
  • D can be a monodentate ligand
  • B can be As or Sb
  • w can be 0, 1, or 2.
  • Exemplary compounds include AsI 3 and sodium stibogluconate (i.e., oxo(A)-gluconato-2,3,4-hydroxodisodic antimony (V) sodium salt).
  • sodium stibogluconate includes Pentostam ® (Glaxo Wellcome Inc., London, England).
  • arsenic-, antimony-, or bismuth-containing polymeric compounds such as -O-As(O - )-O-As(O - )-O-As(O - )-, can be used. These compounds, upon administration to a subject, are hydrolyzed to compounds of formula (I).
  • the term "monodentate ligand” refers to an ion that has one point at which it attaches to atom B, and may have a valence of one or two.
  • Examples of a monodentate monovalent ligand include fluoro, chloro, bromo, iodo, cyano, hydroxyl, mercapto, alkoxyl, thioalkoxyl, nitrate, perchlorate, carboxylate, amide, trialkylsilyl, isocyanate, isothiocyanate, imidazole, phosphate, and alkyl- or aryl-sulfonate.
  • Examples of a monodentate divalent ligand include oxygen, sulfur, selenide, telluride, and sulfate.
  • the term "bidentate ligand” refers to a compound that possesses two points at which it attaches to atom B. Examples of bidentate ligands include tartrate and polyol-type ligand (e.g., meglumine or carbohydrates).
  • the term "tridentate ligand” refers to a compound that possesses three points at which it attaches to atom B. Examples of tridentate ligands include citrate and polyol-type ligand (e.g., meglumine or carbohydrates).
  • alkyl refers to a straight-chained or branched alkyl group containing I to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, and n-pentyl.
  • alkoxyl refers to a straight-chained or branched group containing 1 to 6 carbon atoms and an oxygen radical.
  • aryl refers to a hydrocarbon ring system having at least one aromatic ring. Examples of aryl moieties include phenyl, naphthyl, and pyrenyl.
  • the method described above is treating infection by a flaviviridae virus, which can be hepatitis C virus, GB virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, or yellow fever virus.
  • a flaviviridae virus which can be hepatitis C virus, GB virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, or yellow fever virus.
  • treating infection includes use of a compound described above for preventing or treating cirrhosis, hepatocellular carcinoma, or other disease states secondary to flaviviridae infection.
  • a subject in need of such treatment can be identified by a health care professional based on the results of any diagnostic method.
  • the subject can be concurrently administered with an effective amount of ribavirin. It can also be concurrently administered with an effective amount of interferon ⁇ . Further, it can be concurrently administered with an effective amount of interferon ⁇ and ribavirin.
  • Ribavirin is 1- ⁇ -D-ribofuranosyl-1,2,4-triazole-3-carboximide, which can be chemically synthesized.
  • Commercially available ribavirins include VILONA3 (ICN Pharmaceuticals, Canada).
  • Examples of interferon ⁇ which can be in unmodified or modified form, include interferon ⁇ -2a, interferon ⁇ -2b, and Pegylated interferon a (e.g., peginterferon ⁇ -2b).
  • interferons include ROFERON ® -A (Roche Pharmaceuticals, Nutley, NJ), INTRON A3 (Schering Corporation, Kenilworth, NJ), INFERGEN ® (Amgen, Thousand Oaks, CA), and WELLFERON3 (Glaxo Wellcome Inc., Research Triangle Park, NC).
  • interferons can be prepared by a synthetic method (e.g., by using a peptide synthesizer) followed by proper folding in vitro, or by a recombinant method using E. coli, yeast, insect cells, plant cells, or mammalian cells.
  • a combination of ribavirin and interferon ⁇ -2b is sold as REBETRON3 by Schering Corporation.
  • Both interferon ⁇ and ribavirin can be also in the form of a pharmaceutical acceptable salt.
  • a salt for example, can be formed between a negatively charged substituent (e.g., carboxylate) on interferon ⁇ or ribavirin and a cation.
  • Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion.
  • a positively charged substituent e.g., amino
  • Suitable counterions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, or acetate.
  • ribavirin is administered orally with an amount of from 10 mg/day to about 50,000 mg/day.
  • Interferon ⁇ is administered parenterally (e.g., intramusclar or intracutaneous) with an amount of from about 0.1 to about 50 million International Units per week (e.g., three times per week).
  • the compound can be administered orally, parenterally (e.g., intramuscular, intracutaneous, or intrathecal), by inhalation spray, or via an implanted reservoir.
  • this invention features a pharmaceutical composition that includes a compound described above and ribavirin, wherein each of the compound and the ribavirin is in an effective amount for treating flaviviridae infection.
  • This invention also features a pharmaceutical composition that includes one or more of the aforementioned compounds and interferon ⁇ , optionally, ribavirin, wherein each of the compound, the interferon ⁇ , and the ribavirin, if present, is in an effective amount for treating flaviviridae infection.
  • a pharmaceutical composition that includes a compound described above, wherein the compound has not been known to possess a pharmaceutical activity, and is in an effective amount for treating flaviviridae infection.
  • this invention features a kit for treating infection by a flaviviridae virus.
  • the kit includes one of the aforementioned compounds and ribavirin, wherein each of the compound and the ribavirin is in an effective amount for treating flaviviridae infection.
  • the kit includes the compound and interferon ⁇ , or the compound, interferon ⁇ , and ribavirin, wherein each of the compound, the interferon ⁇ , and the ribavirin is in an effective amount for treating flaviviridae infection.
  • This invention relates to use of a compound of the formula described in Summary for the preparation of a therapeutic agent for treating flaviviridae infection.
  • the compound includes one or more arsenic, antimony, or bismuth atoms in one molecule.
  • An exemplary compound is arsenic trioxide, which has been known as an antineoplastic agent.
  • arsenic trioxide was approved by the U.S. Food and Drug Administration (FDA) in the fall of 2000 for treating acute promyelocytic leukemia in patients not responding to other medication regimens. In 2001, the FDA also approved injection of arsenic trioxide for treating both chronic myeloid leukemia and acute myelocytic leukemia.
  • Arsenic trioxide is commercially available as a drug.
  • sodium stibogluconate (Pentostam ® ) has been used virtually as the only substance in English-speaking East Africa for the treatment of kala-azar and post-kala-azardermal leishmaniasis.
  • These arsenic-, antimony- or bismuth-containing compounds described above can be synthesized from inorganic chemicals. See, e.g., Handbook of Preparative Inorganic Chemistry Vol. 1, G. Brauer Ed, 2 nd ed., (1963) Academic Press, New York, p 600.
  • a method for treating flaviviridae infection is disclosed with one or more aforementioned compounds (or pharmaceutically acceptable derivatives thereof, or compositions including the compounds or derivatives thereof).
  • the method includes administering to a subject in need thereof an effective amount of the compound.
  • the subject is concurrently administered with an effective amount of interferon ⁇ , or ribavirin, or interferon ⁇ and ribavirin.
  • treating is defined as the application or administration of a composition including the compound described above, alone or in combination with interferon ⁇ , or ribavirin, or interferon ⁇ and ribavirin, to a subject, who has flaviviridae infection, a symptom of flaviviridae infection, a disease state secondary to flaviviridae infection, or a predisposition toward flaviviridae infection, with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate the infection, the symptom of the infection, the disease state secondary to the infection, or the predisposition toward the infection.
  • An effective amount is the amount of the aforementioned compound, interferon ⁇ , or ribavirin (the compound alone or in combination with interferon ⁇ or ribavirin, or in combination with both interferon ⁇ and ribavirin), which, upon administration to a subject in need thereof, is required to confer therapeutic effect on the subject.
  • An effective amount of the compound can range from about 0.001 mg/Kg to about 1000 mg/Kg.
  • An effective amount of interferon ⁇ can range from about 0.1 to about 50 million International Units (IU) per week.
  • An effective amount of ribavirin can range from about 10 mg/day to about 50,000 mg/day. Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other agents for treating flaviviridae infection, such as ursodeoxycholic acid.
  • the compound described above can be administered orally, parenterally, by inhalation spray, or via an implanted reservoir; and interferon ⁇ can be administered parenterally.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection, as well as various infusion techniques, such as intrahepatic infusion.
  • a composition for oral administration can be any orally acceptable dosage form including tablets, capsules, emulsions and aqueous suspensions, dispersions and solutions.
  • Commonly used carriers for tablets include lactose and corn starch.
  • Lubricating agents, such as magnesium stearate, are also typically added to tablets.
  • useful diluents include lactose and dried corn starch.
  • a sterile injectable composition e.g., aqueous or oleaginous suspension
  • a sterile injectable composition can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • suitable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di-glycerides).
  • Fatty acids such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.
  • An inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • a pharmaceutically acceptable carrier is routinely used with the aforementioned compound or interferon ⁇ .
  • a carrier is compatible with the active ingredient of the formulation and not deleterious to the subject to be treated.
  • solubilizing agents such as cyclodextrins (which form specific, more soluble complexes with the compound), can be utilized as pharmaceutical excipients for delivery of the compound.
  • a capsule for encasing the compound such as arsenic trioxide is also a carrier.
  • examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow # 10.
  • An in vitro assay can be used to evaluate the efficacy of the compound described above, or together with interferon ⁇ (in various forms), in inhibiting hepatitis C virus RNA replication.
  • the efficacy and dosages can be further evaluated in animal studies with procedures well known in the art. Examples of efficacy-evaluating methods are provided below.
  • Huh-7 cells containing HCV sub-genomic replicons were provided by Apath, LLC. (St. Louis, MO). See, e.g., Blight et al . (2000) Science 290:1972-4. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) in a humidified atmosphere containing 5% of CO 2. The medium also contained 1 mg/mL G418 (an aminoglycosidic antibiotic agent obtained from Promega, WI) as a selective pressure for the maintenance of HCV replicon's RNA replication. See, e.g., Lohmann et al .
  • DMEM Dulbecco's modified Eagle's medium
  • FCS heat-inactivated fetal calf serum
  • IFN- ⁇ Human interferon ⁇
  • As 2 O 3 arsenic trioxide
  • Sb 2 O 3 antimony trioxide
  • IFN- ⁇ was stored at -20°C in small aliquots.
  • a stock solution of As 2 O 3 (10 mM) was prepared by dissolving As 2 O 3 powder in 1 N NaOH.
  • Stock solutions of Sb 2 O 3 (10 mM) or Bi 2 O 3 (10 mM) were prepared by dissolving Sb 2 O 3 or Bi 2 O 3 powders in HCl/H 2 O (1:1).
  • Stock solutions of PAT (50 mM) or NaAsO 2 (10 mM) were prepared by dissolving PAT or NaAsO 2 powders in H 2 O.
  • Cells were treated with As 2 O 3 (0 to 1 ⁇ M), with Sb 2 O 3 at various concentrations (0 to 5 ⁇ M), or with As 2 O 3 (0 to 100 nM) in combination with IFN- ⁇ (0 to 1 IU/mL). Then, cells were harvested after 24 and/or 72 hr of the treatment. Subsequently, total cellular RNA's were extracted and quantified as follows.
  • RNA of cells was extracted using RNeasy Mini Kit (QIAGEN). The concentration of total RNA was measured by absorption at 260 nm. Total RNA (1 ⁇ g) was then mixed with 50 picomoles of HCV specific reverse primer: A9412, 5'-GATGGC CTATTG GCCTGG AGGGG-3' (Lohmann et al . (2001) J Virol . 75:1437-49). RNA was added with RNase-free ddH 2 O to a final volume of 10.5 ⁇ L and denatured for 10 min at 65°C.
  • RT Reverse transcription
  • Quantitative PCR was carried out by LightCycler-DNA Master using SYBR Green I for real-time detection of double-stranded DNA (dsDNA) (Roche Diagnostics GmbH, Mannheim, Germany). The intensity of fluorescence was recorded versus PCR cycle numbers and the relative amounts of HCV RNA were calculated corresponding to the standard curve.
  • the standard curve was derived from five serial dilutions of reference DNA, the corresponding cDNA of HCV RNA, under criteria recommended by the supplier.
  • HCV RNA and a house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or actin were determined by Q-PCR utilizing the following primer sets:
  • PCR was performed as follows: (i) denaturing conditions: 95°C, 60 sec; (ii) amplification conditions: ramp to 95°C, 0 sec; 64°C, 10 sec; 72°C, 10 sec.
  • the specificity of an amplification reaction was determined by performing a melting curve analysis with a temperature range from 65°C to 95°C.
  • HCV RNA copy numbers were divided by their corresponding GADPH copy numbers in the same sample for normalization. Effect of drug treatments on HCV RNA replication was represented by "relative copy number,” which was obtained by comparing HCV RNA copy numbers with drug treatment to HCV RNA copy numbers without drug treatment. The latter was designated as 100%. All measurements were performed in duplicates or triplicates to reach statistical significance.
  • Huh-7 cells containing HCV sub-genomic replicons (Ava.5) were maintained in DMEM supplemented with 10% FCS in a humidified atmosphere containing 5% of CO 2.
  • Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), as described in Malich et al . (1997) Toxicology 124:179-92.
  • MTS in the presence of phenazine methosulfate (PMS), produced a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • MTS and PMS both in powdery form, were purchased from Sigma or Promega. MTS was dissolved in PBS (2 mg/mL) and PMS (0.38 mg/mL) was dissolved in ddH 2 O as stock solutions. Cells cultured in a 96-well microtiter plate were treated with As 2 O 3 at various concentrations. Prior to the assay, a 2 mL MTS/PMS solution containing MTS and PMS (20:1) was added to the cells with 8 mL of phenol red-free DMEM. The cell culture medium was then aspirated off, washed with PBS, and 100 ⁇ L of the MTS/PMS mixture was added into each well. After incubation at room temperature for 2 hr, optical densities of each sample were measured at OD 490 with a 96-well microtiter plate reader. Data were obtained with six repeats for each treatment with drugs.
  • Cyotoxicity of As 2 O 3 on the HCV sub-genomic replicon cell line was determined by the just-described MTS assay.
  • the ICso values i.e., the concentration of As 2 O 3 which causes 50% cell death after the cells are treated with As 2 O 3
  • the EC 50 i.e., the effective concentration of As 2 O 3 which inhibits 50% of HCV RNA replication as determined by Q-PCR described above
  • HCV replication inhibition assay was performed using an Epstein-Barr virus-based extrachromosomal replication system as described in Yeh et al . (2001, J. of Virology 75:11017-11024). Results show that 300 nM As 2 O 3 was able to inhibit the replication completely (data not shown).
  • EC 50 and IC 50 values for 24 hr treatment with sodium arsenite, arsenic iodide, antimony trioxide, potassium antimony tartrate, and bismuth trioxide were also determined using Ava5 cells (Table 4). Table 4. Anti-HCV effects.
  • Name Formula EC 50 a IC 50 b Note Arsenic trioxide As 2 O 3 200 nM 6 ⁇ M Sodium arsenite NaAsO 2 250 nM 10 ⁇ M Arsenic iodide AsI 3 1 ⁇ M 10 ⁇ M Antimony trioxide Sb 2 O 3 800 nM 12.5 ⁇ M Potassium antimony tartrate (PAT) C 8 H 4 O 12 Sb 2 K 2 N.A.
  • EC so Ava5 cells were harvested after 24 hours of drug treatment. Subsequently, total cellular RNA was extracted and quantified. The levels of HCV RNA (positive-strand) and the control mRNA of a housekeeping gene, GADPH or actin, were measured by Q-PCR. HCV RNA levels in each experiment were divided by respective GADPH and actin mRNA levels to obtain normalized HCV RNA levels.
  • b IC 50 Cytotoxicity of compounds on Ava.5 cells was determined by MTS assay after treatment for 24 hours.
  • c N.A. not available.

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